Utilizing Mutant Strains and Characterization of Factors Sufficient for Gaba Utilization As a Carbon and Nitrogen Source
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ISOLATION OF y-AMINOBUTYRATE (GABA) UTILIZING MUTANT STRAINS AND CHARACTERIZATION OF FACTORS SUFFICIENT FOR GABA UTILIZATION AS A CARBON AND NITROGEN SOURCE. ISOLATION OF y-AMINOBUTYRATE (GADA) UTILIZING MUTANT STRAINS AND CHARACTERIZATION OF FACTORS SUFFICIENT FOR GADA UTILIZATION AS A CARBON AND NITROGEN SOURCE By SAROSH YOUSUF, B.Sc. (Hons.) A Thesis Submitted to the School of Graduate Studies in Partial Fulfilment of the Requirements for the Degree Master of Science McMaster University © Copyright by Sarosh Yousuf, January 2001 MASTER OF SCIENCE McMaster University (Biology) Hamilton, Ontario TITLE: Isolation ofy-aminobutyrate (GABA) utilizing mutant strains and characterization of factors sufficient for GABA utilization as a carbon and nitrogen source. AUTHOR: Sarosh Yousut: RSc. (Hons) (Shippensburg University) SUPERVISOR: Dr. H.E. Schellhorn NUMBER OF PAGES: xi, 94 ii Abstract We have previously found that the gab operon ofEscherichia coli is RpoS regulated suggesting that it plays an important role during stationary phase growth. The specific role of the gab operon is metabolism of y-aminobutyrate (GABA). GAB~ a four carbon amino acid, is the product ofthe glutamate decarboxylase reaction in E. coli and can serve as a source of carbon and nitrogen for members ofthe Enterobacteriaceae. Interesting]y, we found that we could isolate GABA utilizing mutants even when they carried mutations that were predicted to abolish gab function. However, we were only able to isolate GABA utilizing mutants in the rpos*" background. This suggests two additional features of GABA metabolism in E. coli. One, that the gab operon in Escherichia coli is indeed dispensable for GABA utilization. Secondly, other RpoS regulated functions, independent ofgab-encoded functions, may allow the cell to metabolize GABA Finally, we have also investigated the inducing effects of GABA on gab expression in minimal and rich media and the role ofglutamate in osmo-tolerance and acid adaptation. iii ACKNOWLEDGMENTS Firstly, I would like to thank my supervisor, Dr. H.E. Schellhorn for providing me with the opportunity to study at McMaster University in pursuing a M.Sc. degree in his laboratory. I am grateful to him for his constructive criticism, patience, and overall support throughout the course of this project. I am also grateful to Dr. Dick Morton for his comments and suggestions during the thesis write-up process and for sitting on my committee. My sincere thanks to my fellow lab members, both past and present, namely Jonathon P. Audia, Chantalle K. D' Souza, Lily Chang, Galen Chen, Dr. Rouha Kasra and ofcourse all the summer students for all their technical advice, support and the laughs. Many thanks to all the friends I have made in the Biology Department. All this has helped make my stay here at McMaster University an enjoyable and memorable one. Finally, I would like to thank my parents for their unconditional love and their continued support in every way possible. Also, I would like to thank God for putting me in the position to make something out of my life. iv TABLE OF CONTENTS Page Abstract iii Acknowledgments iv Table of Contents v List of Abbreviations Vlll List ofTables ix List of Figures X CHAPTER!. INTRODUCTION. 1.1 Overview 1 I .2 RpoS as a regulator 2 1.3 GABA: an inhibitory neurotransmitter in eukaryotes 4 1.4 The Gab Operon 5 1.4.1 Organization of the gab cluster 5 1.4.2 Amino acid homologies of the gab gene products 7 1.5 Pathways Involved in GABA utilization 9 1.5.1 GABA transport in E. coli 9 1.5.2 GABAmetabolisminE. coli 10 1.6 Response of the gab operon to catabolite repression 12 I.7 Pathway of arginine catabolism 15 1.8 Project Outlines and Objectives 15 v CHAPTER2. MATERIALS AND METHODS. 2.1 Bacterial Strains 24 2.2 Chemicals and Media 24 2.3 Growth Conditions 24 2.4 Assay for (3-galactosidase 24 2.5 PCR amplification and sequence analysis 25 2.6 Isolation of GABA utilizing mutants 26 2. 7 Genetic methods 28 2.8 Isolation ofGABA utilizing mutants from fusion strains 28 2.9 Calculation of mutational frequency 29 2.10 Preparation of Conditioned Medium 29 2.11 GABA induction ofgab expression in Minimal Medium 30 2.12 Acid Adaptation Test 30 2.13 Osmoprotection Test 31 CHAPTER3. RESULTS 3.1 GABA utilizing mutants obtained 37 3.2 (3-galactosidase assay using transduced GABA mutants 38 3.3 Sequence analysis of the gab promoter in the GABA mutants 39 3.4 Dispensability of the gab operon for GABA utilization 40 vi 3.5 Putative inducers ofgab expression 42 3.5.1 Conditioned Medium 42 3.5.2 LB media supplemented with GABA (0.2%) 43 3.5.3 Minimal media supplemented with GABA (0.2%) 44 3.6 Role of glutamate in acid adaptation and osmotolerance 44 CHAPTER4. DISCUSSION 79 4.1 References 87 vii List of Abbreviations ADC Arginine decarboxylase-initiated pathway AST Arginine succinyltransferase pathway ea2+ Calcium csiD Carbon-starvation inducible gene D CNS Central nervous system cAMP-CRP cyclic adenosine monophosphate receptor protein oc Degrees Celsius DNA Deoxyribonucleic acid dNTP 2'-deoxynucleotide-5' -triphosphate GABA y-aminobutyrate Group I (CN) Carbon and nitrogen utilizing GABA mutant Group II (N) Nitrogen utilizing GABA mutant Group III (C) Carbon utilizing GABA mutant LB Luria-Bertani LXS Luria-Bertani-5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside Streptomycin J.Lg Microgram J.Ll Micro litre mg Milligram ml Milliliter mM Millimolar J.Lm Micro molar MF Mutational frequency ng Nanogram nm Nanometer OD Optical density 0/N Overnight ONPG o-nitrophenyl-P-D-galactopyranoside PCR Polymerase chain reaction REP Repetitive extragenic palindromic elements rpm Revolutions per minute RNA Ribonucleic acid uv Ultraviolet X-Gal 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside viii List of Tables Page CHAPTER I Table I. Members of the RpoS regulon in E. coli. 17 CHAPTER3 Table 2a. E. coli strains and bacteriophage used in this study. 47 Table 2b. E. coli strains and bacteriophage used in this study. 49 Table 3. Analysis ofGABA mutants obtained for utilization ofGABA as a carbon and/or nitrogen source. 50 Table 4. GABA mutants obtained from wild type fusion backgrounds. 51 Table 5. Over-expressing GABA mutants obtained. 52 Table 6. Expression ofgab genes in response to conditioned medium. 53 Table 7. Expression of gab genes in response to GABA (0.2%). 54 Table 8. Growth of wild type and GABA mutant strains on various weak acids in the presence and absence ofGABA 55 Table 9. Growth of wild type and GABA mutant strains on varying concentrations of salt in the presence and absence of glutamate. 56 ix List of Figures Page CHAPTER! Figure 1. Schematic representation of the gab region. 18 Figure 2. The pathway of GABA degradation in E. coli. 20 Figure 3. The arginine decarboxylase-initiated (ADC) pathway of arginine catabolism. 22 CHAPTER2 Figure 4. Strategy employed to isolate three different groups of GABA utilizing mutant strains directly from the wild type (MC4100) background. 33 Figure 5. Schematic representation of the strategy used to isolate GABA utilizing mutants directly from strains carrying lacZ fusions to members of the gab operon. 35 CHAPTER3 Figure 6a. Expression ofgabP in wild type strains 57 Figure 6b. Expression ofgabP in CN GABA utilizing mutant strains 59 Figure 6c. Expression of gabP inN GABA utilizing mutant strains 61 Figure 6d. Expression ofgabP in C GABA utilizing mutant strains 63 Figure 7. Clustal W sequence alignment results ofPCR amplified csiD promoter region in wild type (MC41 00) strain and three classes of GABA utilizing mutant strains generated from MC41 00. 65 Figure 8. Replica plate pattern of wild type strain HSI057° 0 (rpoS, gabD-lacZ), its rpoS derivative HS I 057p , and Group I (CN) GABA utilizing mutants (HS1903a-c) generated from the wild type rpoS fusion background only. 67 X Figure 9. Expression of gabD in wild type and "over-expressing" GABA utilizing mutant strains. 69 Figure 10. Conditioned medium as an inducer of gab expression. 71 Figure 11. GABA as an inducer ofgab expression in LB media 74 Figure 12. GABA as an inducer ofgab expression in minimal media 77 XI CHAPTERI. llffRODUCTION 1.1 Overview Adaptation to different environmental stresses is critical for the survival of microorganisms. In their natural habitats, bacteria frequently encounter suboptimal growth conditions and in order to cope with these unfavourable circumstances, most microbes have developed complex stress response mechanisms. For instance, gram positive bacteria such as Bacillus subtilis respond to stresses by differentiating into highly resistant spores, while gram-negative species such as Escherichia coli and Salmonella typhimurium make use of stationary phase response mechanisms (Hengge-Aronis, 1996b). Stationary phase cultures are easily distinguishable from those existing in exponential phase growth. This is because cells go from a state ofmaximal cell division and proliferation in exponential phase (generation time for E. coli in LB media is 25 minutes) to one of minimal cell growth and dormancy associated with stationary phase. Interestingly, this shift from exponential to stationary phase is accompanied by many changes in cellular physiology and morphology ofthe bacterial cultures with the purpose to offer longevity and increased resistance to the stresses associated with post exponential phase growth. For instance, E. coli cells, normally rod-shaped during exponential phase growth, assume a smaller and more spherical shape in stationary phase (Lange et al., 1991a; Reeve et al., 1984). Moreover, the cytoplasm of the cells 2 condenses, the volume ofthe periplasm increases (Lange et al., 1991a; Reeve et al., 1984), the cell wall becomes cross linked and there is an increase in the degree ofDNA supercoiling (Balke et aL, 1987). 1.2 RpoS as a regulator The end of exponential phase is marked by increased expression of genes which seem to help bacteria combat multiple stresses.